IL43343A

IL43343A - Production of plasminogen activator

Info

Publication number: IL43343A
Application number: IL43343A
Authority: IL
Original assignee: Lilly Co Eli
Priority date: 1972-10-24
Filing date: 1973-09-30
Publication date: 1976-03-31
Also published as: GB1443189A; SE392618B; CH589142A5; FR2203876B1; BE806323A; JPS4975794A; NL7314579A; SE7609880L; FR2203876A1; IL43343A0; DE2353318A1

Claims

We claim:

1. Λ process for the preparation of plasminogen activator which comprises : a) culturing plasminogen activator producing mammalian cells in an aqueous nutrient tissue culture medium containing assimilable sources of nitrogen, carbon and inorganic salts to obtain a viable cell culture; b) culturing said viable cell culture in an aqueous nutrient tissue culture medium containing between 0.1 and 10 mcg/ml . of an alkaloid selected from the group consisting of colchicine, colchamide, desacetylcolchicine , desacetamidocolchicine , cyanocolchicine , vinblastine and vincristine until a substantial amount of plasminogen activator is present in said medium; and c) recovering the plasminogen activator from said tissue culture medium.

2. The process of claim 1 wherein the mammalian cells are cultured in the presence of an inert solid support to form a viable culture of confluent cells on said support.

3. The process of claim 1 wherein the mammalian cells are cultured to maximum population density in a viable, submerged cell suspension 'culture.

4. The process of any of claims 1, 2 or 3 wherein the mammalian cells are mammalian kidney cells.

5. The process of any of claims 1-4 in which the mammalian cells are porcine kidney cells PK (15) ; ATCC No. CCL 33 PK (15) . 0

6. The process of any of claims 1-4 in which the mammalian cells are porcine kidney cells LLC-PK, (Hull), ATCC No. CL 101.

7. The process of any of claims 1-4 in which the mammalian cells are rabbit kidney cells (Hull) , ATCC No. CCL 106.

8. The process of any of claims 1-4 in which the mammalian cells are rhesus monkey kidney cells LLC-MI<2 (Hull) , ATCC No. CCL 7.

9. The process of any of claims 1-4 in which the mammalian cells are rhesus monkey kidney cells LLC-MI^/ ATCC No. CCL 7.1.

10. The process of any of claims 1-4 in which the mammalian cells are porcine kidney cells LLC-PK^, ATCC No. CL 101.1.

11. The process of any of claims 1-10 wherein the alkaloid is colchicine.

12. The process of any of claims 1-10 wherein the alkaloid is colchamide.

13. The process of any of claims 1-10 wherein the alkaloid is vinblastine.

14. The process of any of claims 1-10 wherein the alkaloid is vincristine.

15. The process of claim 1 wherein a purified plasminogen activator is recovered from an aqueous nutrient tissue culture medium by. a) separating insolubles from the aqueous tissue medium containing plasminogen activator ; b) diluting the separated medium with distilled water to adjust the specific conductance of said medium to 8 millir.hos or less; c) contacting said plasminogen activator containing medium for between 1 and 24 hours with between 3 and 12 g. of hydroxyapatite per liter of medium; d) separating the hydroxyapatite containing adsorbed plasminogen activator from said medium; e) washing said hydroxyapatite; f) eluting said plasminogen activator from the washed hydroxyapatite with 0.7 M phosphate buffer at pH 6.8; g) adding to the plasminogen activator containing eluate a water soluble inorganic salt in an amount sufficient to achieve a salt concentration of appro imately 20 percent of saturation ; h) separating the precipitated impurities from said eluate; i) adding to said eluate an additional amount of said salt to achieve a salt concentration of between approximately 45 and 90 percent of saturation; j) separating the precipitated plasminogen activator from said salt containing eluate; k) dissolving the plasminogen activator in phosphate buffer having a molarity of between approximately 0.001 and 0.005 and dialyzing said buffer solution; 1) contacting the dialyzed solution of plasminogen activator with diethylaminoethyl cellulose adsorbent; m) eluting the adsorbed plasminogen activator from said adsorbent with 0.005 molar phosphate buffer; n) adding to the plasminogen activator containing eluate a water soluble inorganic salt in an amount sufficient to achieve a salt concentration of approximately 75 percent of saturation; o) separating the precipitate of plasminogen activator; 10 p) dissolving the plasminogen activator in 0.1 tris-hydroxymethylaminomethane : 0.1 M potassium chloride buffer, pll 8.0 and dialyzing the buffer against said buffer alone; q) contacting the dialyzed buffer solution with a modified dextran gel; r) eluting the adsorbed plasminogen activator from said gel with 0.1 M tris-hydroxymethylamino- methane:0.1 14 potassium chloride, pH 8.0 buffer; and 20 s) lyophilizing the plasminogen activator containing eluate to obtain in a purified solid form the plasminogen activator.

16. The method of claim 15, in which the water soluble inorganic salt is ammonium sulfate.

17. The process of preparing plasminogen activator substantially as herein described with particular reference to any one of the specific examples.

18. The process of preparing plasminogen activator substantially as herein described with particular reference 30 to any of specific Examples 1-4.

19. The process of preparing plasminogen activator substantially as herein described v/ith particular reference to Example 5. S. HOROWITZ & CO. AGENTS FOR APPLICANTS